c-Jun amino-terminal kinases (JNKs) and mitogen-activated protein kinases (MAPKs) are closely related; however, they are independently regulated by a variety of environmental stimuli. Although molecules linking growth factor receptors to MAPKs have been recently identified, little is known about pathways controlling JNK activation. Here, we show that in COS-7 cells, activated Ras effectively stimulates MAPK but poorly induces JNK activity. In contrast, mutationally activated Rac1 and Cdc42 GTPases potently activate JNK without affecting MAPK, and oncogenic guanine nucleotide exchange factors for these Rho-like proteins selectively stimulate JNK activity. Furthermore, expression of inhibitory molecules for Rho-related GTPases and dominant negative mutants of Rac1 and Cdc42 block JNK activation by oncogenic exchange factors or after induction by inflammatory cytokines and growth factors. Taken together, these findings strongly support a critical role for Rac1 and Cdc42 in controlling the JNK signaling pathway.

The oncogenic protein Vav harbours a complex array of structural motifs, including leucine-rich, Dbl-homology, pleckstrin-homology, zinc-finger, SH2 and SH3 domains. Upon stimulation by antigens or mitogens, Vav becomes phosphorylated on key tyrosine residues and associates with other signalling proteins, including the mitogen receptors Zap-70 (ref. 6), Vap-1 (ref. 5) and Slp-76 (ref. 7). Disruption of the vav locus by homologous recombination causes severe defects in signalling by primary antigen receptors, leading to abnormal lymphocyte proliferation and lymphopenia. Despite the importance of Vav cell signalling, the function of this protein remains unknown. Here we show that tyrosine-phosphorylated Vav, but not the non-phosphorylated protein, catalyses GDP/GTP exchange on Rac-1, a protein implicated in cell proliferation and cytoskeletal organization, causing this GTPase to switch from its inactive to its active state. Transfection experiments also show that phosphorylation of Vav on tyrosine residues leads to nucleotide exchange on Rac-1 in vivo and stimulates c-Jun kinase, a downstream element in the signalling pathway involving this GTPase. Our results have identified a function for Vav and define a mechanism in which engaged membrane receptors activate its signalling pathway.

The tyrosine kinase class of receptors induces mitogen-activated protein kinase (MAPK) activation through the sequential interaction of the signaling proteins Grb2, Sos, Ras, Raf, and MEK. Receptors coupled to heterotrimeric guanine triphosphate-binding protein (G protein) stimulate MAPK through Gbetagamma subunits, but the subsequent intervening molecules are still poorly defined. Overexpression of phosphoinositide 3-kinase gamma (PI3Kgamma) in COS-7 cells activated MAPK in a Gbetagamma-dependent fashion, and expression of a catalytically inactive mutant of PI3Kgamma abolished the stimulation of MAPK by Gbetagamma or in response to stimulation of muscarinic (m2) G protein-coupled receptors. Signaling from PI3Kgamma to MAPK appears to require a tyrosine kinase, Shc, Grb2, Sos, Ras, and Raf. These findings indicate that PI3Kgamma mediates Gbetagamma-dependent regulation of the MAPK signaling pathway.

Mitogen-activated protein kinases, MAP kinases or ERKs (extracellular signal-regulated kinases) are rapidly stimulated by growth-promoting factors acting on a variety of cell-surface receptors. In turn, ERKs phosphorylate and regulate key intracellular enzymes and transcription factors involved in the control of cellular proliferation. The tyrosine-kinase class of growth-factor receptors transmits signals to ERKs in a multistep process that involves Ras and a limited number of defined molecules. In contrast, ERK activation by G-protein-coupled receptors is poorly understood, as is the role of ras in this signalling pathway. We have explored in COS-7 cells the mechanism of ERKs activation by m1 and m2 muscarinic receptors, typical examples of receptors coupled through Gq proteins to induce phosphatidylinositol hydrolysis and to G(i) proteins to inhibit adenylyl cyclase, respectively. Here we present evidence that ERK activation is mediated by beta gamma subunits of heterotrimeric G proteins acting on a ras-dependent pathway.

The four receptor tyrosine kinases of the ErbB family play essential roles in several physiological processes and have also been implicated in tumor generation and/or progression. Activation of ErbB1/EGFR is mainly triggered by epidermal growth factor (EGF) and other related ligands, while activation of ErbB2, ErbB3, and ErbB4 receptors occurs by binding to another set of EGF-like ligands termed neuregulins (NRGs). Here we show that the Erk5 mitogen-activated protein kinase (MAPK) pathway participates in NRG signal transduction. In MCF7 cells, NRG activated Erk5 in a time-and dose-dependent fashion. The action of NRG on Erk5 was dependent on the kinase activity of ErbB receptors but was independent of Ras. Expression in MCF7 cells of a dominant negative form of Erk5 resulted in a significant decrease in NRG-induced proliferation of MCF7 cells. Analysis of Erk5 in several human tumor cell lines indicated that a constitutively active form of this kinase was present in the BT474 and SKBR3 cell lines, which also expressed activated forms of ErbB2, ErbB3, and ErbB4. Treatments aimed at decreasing the activity of these receptors caused Erk5 inactivation, indicating that the active form of Erk5 present in BT474 and SKBR3 cells was due to a persistent positive stimulus originating at the ErbB receptors. In BT474 cells expression of the dominant negative form of Erk5 resulted in reduced proliferation, indicating that in these cells Erk5 was also involved in the control of proliferation. Taken together, these results suggest that Erk5 may play a role in the regulation of cell proliferation by NRG receptors and indicate that constitutively active NRG receptors may induce proliferative responses in cancer cells through this MAPK pathway.Receptor tyrosine kinases of the ErbB family play essential roles in several physiological processes, such as cell growth (11,36,66), differentiation, and tissue development (8,55,61), and have been implicated in pathological processes, such as tumor generation and/or progression (36,66). This family comprises four structurally related transmembrane receptors, the epidermal growth factor (EGF) receptor (EGFR or ErbB1/HER1), ErbB2 (neu/HER2), ErbB3 (HER3), and ErbB4 (HER4) (36,66). Activation of ErbB receptors may occur by ligand binding (67,68) or by overexpression of the receptor (36, 57), the latter mechanism being particularly relevant in certain pathologic instances such as cancer (30,(62)(63)(64). Ligand-mediated activation of ErbB receptors occurs by interaction of the ectodomain of these receptors with specific members of the EGF family of ligands (11,48). This family includes EGF, transforming growth factor ␣, amphiregulin, betacellulin, and epiregulin, which preferentially bind to and activate the EGFR (3,48,65). A second group of EGF-like ligands, the neuregulins (NRGs), bind to ErbB3 and ErbB4 (6,38,53). Ligand-induced activation of ErbB receptors is complex and often includes oligomeric interactions between different ErbB receptors (19,54). Thus, upon ligand binding, ErbB recepto...

Sequestration of c-Fos at the nuclear envelope (NE) through interaction with A-type lamins suppresses AP-1–dependent transcription. We show here that c-Fos accumulation within the extraction-resistant nuclear fraction (ERNF) and its interaction with lamin A are reduced and enhanced by gain-of and loss-of ERK1/2 activity, respectively. Moreover, hindering ERK1/2-dependent phosphorylation of c-Fos attenuates its release from the ERNF induced by serum and promotes its interaction with lamin A. Accordingly, serum stimulation rapidly releases preexisting c-Fos from the NE via ERK1/2-dependent phosphorylation, leading to a fast activation of AP-1 before de novo c-Fos synthesis. Moreover, lamin A–null cells exhibit increased AP-1 activity and reduced levels of c-Fos phosphorylation. We also find that active ERK1/2 interacts with lamin A and colocalizes with c-Fos and A-type lamins at the NE. Thus, NE-bound ERK1/2 functions as a molecular switch for rapid mitogen-dependent AP-1 activation through phosphorylation-induced release of preexisting c-Fos from its inhibitory interaction with lamin A/C.

Ras GTPases-H-Ras, N-Ras, and K-Ras 4B/4A-operate as key molecular switches that convey extracellular signals from surface receptors to the interior of the cell, thereby regulating essential processes including proliferation, differentiation, and survival (15,34). It is well known that Ras must be attached to the inner leaflet of the plasma membrane (PM) to be functional (50). This is accomplished by lipidic additions to the protein C terminus (33), which contains the essential signal for localizing Ras to membranes: the CAAX box (where C is cysteine, A is alyphatic amino acid, and X is serine/methionine). This motif undergoes posttranslational modifications that make it more hydrophobic. The cysteine is farnesylated, the AAX sequence is proteolyzed, and the newly C-terminal cysteine is carboxymethylated (50). However, a second signal is necessary for efficiently positioning Ras in the membrane. This is accomplished by palmitoylation of cysteine 181 in N-Ras, and cysteines 181 and 184 in H-Ras. In the case of K-Ras 4B the second signal is attained by a polybasic motif of six lysines (175 to 180) that interacts electrostatically with the negatively charged membrane (24-26).Recently, a new twist has been provided by findings indicating that Ras isoforms are distinctively segregated in different PM microdomains with unique biochemical and physicochemical characteristics, H-Ras can be found in bulk membrane and in lipid rafts, both caveolar and noncaveolar. K-Ras is exclusively located in bulk membrane, whereas N-Ras can only be detected in noncaveolar lipid rafts (35,(38)(39)(40). Furthermore, recent reports indicate that Ras is also present in endomembranes such as endosomes, endoplasmic reticulum (ER), and the Golgi complex (10,37,45). The significance of this distribution seems to go beyond that of a transient event associated to transport and/or recycling. Instead, a pool of Ras appears to reside in these organelles, and therein Ras can productively engage downstream effectors (10,11,37,45). Moreover, at these endomembranes Ras regulation is undertaken by proteins that operate in a location-specific fashion. As such, the guanine nucleotide exchange factor RasGRP specifically regulates Ras activation at the Golgi complex (7, 9), whereas SOS and RasGRF undertake Ras regulation at the ER. Likewise, stimuli such as lysophosphatidic acid preferentially activate the Ras pool at the ER, whereas calcium ionophores are more effective in activating PM Ras (4).The fact that exogenous stimuli activate Ras distinctively depending on its localization and that Ras regulation at different sites requires the participation of specific intermediaries hints at the necessity for a location-specific control. This, in term, may imply that Ras functions at its different sites may not be totally redundant. Thus, a selective activation of Ras at each of its locations could be intended to generate variability in its biochemical and biological outputs. It is known that Ras regulates numerous cellular functions through the activation of an...

K-Ras mutations are frequent in colorectal cancer (CRC), albeit K-Ras is the only Ras isoform that can elicit apoptosis. Here, we show that mutant K-Ras directly binds to the tumor suppressor RASSF1A to activate the apoptotic MST2-LATS1 pathway. In this pathway LATS1 binds to and sequesters the ubiquitin ligase Mdm2 causing stabilization of the tumor suppressor p53 and apoptosis. However, mutant Ras also stimulates autocrine activation of the EGF receptor (EGFR) which counteracts mutant K-Ras-induced apoptosis. Interestingly, this protection requires the wild-type K-Ras allele, which inhibits the MST2 pathway in part via AKT activation. Confirming the pathophysiological relevance of the molecular findings, we find a negative correlation between K-Ras mutation and MST2 expression in human CRC patients and CRC mouse models. The small number of tumors with co-expression of mutant K-Ras and MST2 has elevated apoptosis rates. Thus, in CRC, mutant K-Ras transformation is supported by the wild-type allele.